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Comprehensive transcription terminator atlas for Bacillus subtilis

Nature Microbiology volume 7, pages 1918–1931 (2022)Cite this article

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Abstract

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of ‘hybrid Rho-dependent termination’, wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.

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Fig. 1: In silico profile of classical Rho-dependent terminators.
Fig. 2: In silico profile of intrinsic terminators.
Fig. 3: Rut sites are encoded downstream of Rho-stimulated intrinsic terminators.
Fig. 4: Rho cooperates with NusA and NusG to stimulate intrinsic termination.
Fig. 5: Rho stimulates termination in a wide range of transcription contexts.
Fig. 6: Models of Rho-stimulated intrinsic and hybrid Rho-dependent termination.

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Data availability

Sequencing libraries generated in this study are deposited in Gene Expression Omnibus (GEO) (accession entry GSE188366). Previously published datasets for nusAdep and ΔnusG strains can be found at GEO with accession code GSE154522. Source data are provided with this paper.

Code availability

All scripts used to identify total 3’ ends from Term-seq data and calculate their termination efficiencies are archived on GitHub (https://github.com/zfmandell/Term-seq/releases/tag/v1.0).

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Acknowledgements

This work was supported by National Institutes of Health (NIH) Grant GM098399 (to P.B.), NIH grant GM131860 (to K.S.M.) and the Intramural Research Program of the NIH National Cancer Institute (to M.K.). We thank A. Yakhnin for critically reading the manuscript. Anti-σA antibodies and anti-NusA antibodies were obtained from M. Fujita (University of Houston) and P. Lewis (University of Newcastle), respectively. HRP-conjugated goat anti-rabbit antibody is available from GenScript. All bacterial strains are available from P.B.

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  1. Zachary F. Mandell

    Present address: Department of Molecular Biology and Genetics and Department of Biology, Johns Hopkins University, Baltimore, MD, USA

  2. These authors contributed equally: Zachary F. Mandell, Rishi K. Vishwakarma.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, USA

    Zachary F. Mandell, Rishi K. Vishwakarma, Helen Yakhnin, Katsuhiko S. Murakami & Paul Babitzke

  2. NCI RNA Biology Laboratory, Center for Cancer Research, NCI, Frederick, MD, USA

    Mikhail Kashlev

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Contributions

Z.F.M., M.K. and P.B. conceptualized the project. Z.F.M. and R.K.V. developed the methodology. Z.F.M, R.K.V., K.S.M., M.K. and P.B. conducted formal analyis. Z.F.M., R.K.V. and H.Y. conducted investigations. Z.F.M. wrote the original draft. Z.F.M, R.K.V., H.Y., K.S.M., M.K. and P.B. reviewed and edited the manuscript. K.S.M., M.K. and P.B. secured funding.

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Correspondence to Paul Babitzke.

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Extended data

Extended Data Fig. 1 NusA depletion and benchmarking of terminators.

a. Western blot for all samples used for Term-seq was performed once. Top panel, image after probing for NusA. Bottom panel, image after probing for σA as a loading control. Lanes: 1, purified NusA6His (top panel) or σA (bottom panel); 2, PLBS730 + IPTG (WT); 3, PLBS730 –IPTG (nusAdep); 4, PLBS731 + IPTG (ΔnusG); 5, PLBS731 –IPTG (nusAdep ΔnusG); 6, PLBS890 + IPTG (Δrho); 7, PLBS890 –IPTG (nusAdep Δrho); 8, PLBS891 + IPTG (ΔnusG Δrho); 9, PLBS891 –IPTG (nusAdep ΔnusG Δrho). b. Venn diagram showing the number and overlap of intrinsic terminators identified in PLBS727 (WT in this study) and those identified previously in PLBS730 + IPTG (WT in previous study). c-f. RNA-seq coverage data from WT and Δrho strains across the previously identified classical Rho-dependent terminators for ylaL (c), spoVB (d), rapD (e) and slrA (f). Arrows at the bottom indicate the direction of transcription. Top tracks are the 3’ ends identified by Term-seq across each region.

Source data

Extended Data Fig. 2 A-rich tracts and Rho-stimulated intrinsic termination in vivo.

a. A-rich tract motifs generated for the FI, SA, SG, and SR intrinsic terminator subpopulations. A hierarchical clustering analysis can be found at the top of the motifs. b. IGV screenshot of the region upstream of the tbcS terminator. Top track is the 3’ end identified by Term-seq. Bottom tracks are the RNA-seq coverage data for the WT and Δrho strains. Arrow at the bottom indicates the direction of transcription. c. IGV screenshot of RNA-seq and ribosome profiling data across the transcript containing two sfp pseudogenes. Arrows at the bottom indicate the direction of transcription.

Source data

Extended Data Fig. 3 Verification of intrinsic terminator function in vitro.

a. Single-round in vitro termination assay with the tbcS intrinsic terminator. Experiments were performed with templates containing the WT terminator or those in which the DNA corresponding to the U-rich tract (ΔU) or the U-rich tract and the terminator hairpin (ΔU + HP) was deleted. Experiments were performed in the absence (–) or presence of Rho (R). b. Single-round in vitro termination assay with the yybG intrinsic terminator. Experiments were performed with WT or ΔU + HP templates in the absence (–) or presence of Rho (R), NusA (A) and/or NusG (G). c. Single-round in vitro termination assay with the bstB intrinsic terminator. Experiments were performed with WT, ΔHP or ΔU + HP templates in the absence (–) or presence of Rho (R). These qualitative intrinsic terminator validation experiments were performed once.

Source data

Extended Data Fig. 4 Rho-stimulated intrinsic terminators with antiterminators.

a. IGV screenshot of a genomic window centered around the yybG intrinsic terminator. Top track is the 3’ end identified by Term-seq. Bottom tracks are the RNA-seq coverage data for the WT and Δrho strains. %T in each strain is shown on the right of each track. Arrow at the bottom depicts the direction of transcription. b. Model of the yybG intrinsic terminator. c. Single-round in vitro termination assay with the yybG intrinsic terminator. Experiments were performed in the absence (–) or presence of Rho (R). Positions of terminated (Term) and full-length (FL) transcripts are marked. %T ± standard deviation is shown below each lane. Loss of termination in vitro when the terminator was deleted established that this is an authentic intrinsic terminator (Extended Data Fig. 3b). d-f. same as for panels a-c, except that it is the bstB (yuaE) intrinsic terminator. Loss of termination in vitro when the terminator was deleted established that this is an authentic intrinsic terminator (Extended Data Fig. 3c). g. RNA sequence of the ribD, sfp, tbcS, yybG and bstB leader terminators (inverted arrows). Red sequences can participate in the formation of alternative antiterminator (AT) structures. In vitro transcription experiments were performed 3 times. Values are averages ± standard deviation.

Extended Data Fig. 5 Sequencing data from each strain and Rho ATPase assay.

a. Principal component analysis (PCA) plot of transcriptomics data collected from each Term-seq replicate. b. Bar graph showing the nmol of Pi that was released by Rho in the presence of a polyC transcript and in the absence (–) or presence of BCM. ATPase assays were performed 3 times. Values are averages ± standard deviation.

Supplementary information

Reporting Summary

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Supplementary Table 1

All classical Rho-dependent terminators identified in the wild-type strain.

Supplementary Table 2

All intrinsic terminators identified in the wild-type strain.

Supplementary Table 3

Bacterial strains, oligonucleotides, plasmids and P values.

Supplementary Table 4

Analysis of rut sites at Rho-stimulated intrinsic terminators and all intrinsic terminators identified in the wild-type strain and expression data for all strains.

Supplementary Table 5

Results of differential expression analysis for all mutant strains compared to the wild-type strain.

Source data

Source Data Fig. 1

Unprocessed gels for Fig. 4.

Source Data Fig. 2

Unprocessed gels for Fig. 5.

Source Data Extended Data Fig. 1

Unprocessed western Blots for Extended Data Fig. 1.

Source Data Extended Data Fig. 2

Unprocessed gels for Extended Data Fig. 3.

Source Data Extended Data Fig. 3

Unprocessed gels for Extended Data Fig. 4.

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Mandell, Z.F., Vishwakarma, R.K., Yakhnin, H. et al. Comprehensive transcription terminator atlas for Bacillus subtilis. Nat Microbiol 7, 1918–1931 (2022). https://doi.org/10.1038/s41564-022-01240-7

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